The long term objective of this proposal is to investigate hypothalamic control of gonadotropin release in the non-human primate, as a model for the human. The goal of this application is to test the hypothesis that LHRH neurons themselves have an endogenous pulse-generating mechanism, but that they are also controlled by input from other neurons and non- neuronal elements/factors including steroid hormones. To test this hypothesis four specific aims are proposed. The first aim is to determine whether LHRH neurons have an endogenous pulse-generating mechanism using a primary cell culture system for LHRH neurons derived from embryonic olfactory placode of the rhesus monkey. The cellular basis of endogenous pulse generation will be studied in primary LHRH neurons by directly measuring LHRH release with a perifusion system, and examining intracellular Ca2+ oscillations. Even if the in vitro experiment proves that the LHRH neuron generates its own secretory pulses, synaptic input from neuropeptide-Y (NPY) neurons and gamma- aminobutyric acid (GABA) neurons may critically modulate LHRH pulsatility in vivo. Moreover, these neurons appear to be responsible for the action of ovarian steroid hormones in the hypothalamus. Therefore, the second, third and fourth aims comprise in vivo experiments which utilize push-pull perfusion in the stalk-median eminence (S-ME) of ovariectomized monkeys. Specifically, the second aim is to examine the pacemaker for the synchronization of LHRH pulses and NPY pulses, in vivo. Normally, NPY release couples with LHRH release with a lead of NPY pulses, while in some situations LHRH pulses can lead NPY pulses. Antisense oligodeoxynucleotides for LHRH and NPY mRNAs will be infused into the S-ME and changes in the pulsatilities of LHRH release and NPY release will be determined. The third aim is to examine the hypothesis that NPY neurons play a critical role in the feedback action of ovarian steroids. Experiments are proposed to block the progesterone-induced LHRH release and NPY release by direct infusion of an antisense oligodeoxynucleotide for NPY mRNA into the S-ME and to induce an LH surge by pulsatile infusion of NPY in animals treated with a subthreshold dose of estrogen. The fourth aim is to examine the role of GABA in pulsatile LHRH release and in the feedback action of ovarian steroids. The causal relation between GABA pulses and LHRH pulses will be determined by direct measurements of GABA and LHRH in perfusates from the S-ME, by applying the GABA receptor antagonist bicuculline, and by blocking GABA synthesis with antisense oligodeoxynucleotides for glutamic acid decarboxylase (GAD67 and GAD65) mRNAs. Abnormalities of the pulse pattern of LH release, and presumably of LHRH release, are associated with reproductive disorders. Results from the proposed project will provide insight into the mechanisms by which the hypothalamus controls gonadotropin release in primates and ultimately will be useful for clinical management.